Many animals have genetically determined left-right (LR) asymmetry of their internal organs. The midline structure of vertebrate embryos has important roles in LR asymmetric development both as the signaling center for LR asymmetry and as a barrier to inappropriate LR signaling across the midline. However, in invertebrates, the functions of the midline in LR asymmetric development are unknown. To elucidate these roles, we studied the involvement of single-minded (sim) in the LR asymmetry of the Drosophila embryonic gut, which develops in a stereotypic, asymmetric manner. sim encodes a bHLH/PAS transcription factor that is required for the development of the ventral midline structure. Here we report that sim was expressed in the midline of the foregut and hindgut primordia. The handedness of the embryonic gut was affected in sim mutant embryos and in embryos overexpressing sim. However, midline-derived events, which involve Slit/Robo and EGFr signaling and direct the development of the tissues adjacent to the midline, did not affect the laterality of this organ, suggesting a crucial role for the midline itself in LR asymmetry. In the sim mutants, the midline structures of the embryonic anal pad were deformed. The mis-expression of sim in the anal-pad primordium induced LR defects. We also found that different portions of the embryonic gut require sim functions at different times for normal LR asymmetry. Our results suggest that the midline structures are involved in the LR asymmetric development of the Drosophila embryonic gut.